ABSTRACT The mitochondrial protein Prohibitin (PHB) is essential for life. Its importance to cellular activities is attested by the fact that deletion of PHB is embryonic lethal in mice and that to date no mutation has been found in the coding region of PHB in any disease conditions, indicating that PHB integrity is essential and that somatic mutation is detrimental. PHB has been shown to be critical for mitochondrial function in stress situations, as it is upregulated in ischemic preconditioning (IPC). In the previous funding period, using neuron specific PHB transgenic mice, we demonstrated that selective neuronal PHB expression leads to remarkable neuroprotection against middle cerebral arterial occlusion (MCAO) induced brain injury. However, how this important protein is functionally regulated in IPC, as well as how it is dysregulated in other neurological conditions, remain surprisingly unknown. In exploring the mechanisms of PHB regulation, we discovered that nitric oxide (NO) is required for both IPC and PHB upregulation. Therefore, we investigated the interaction between NO and PHB and found that NO modifies PHB post-translationally, through protein s-nitrosylation. In this renewal application, we propose to study the effects of PHB S- nitrosylation and the mechanisms underlying functional regulation of PHB by NO. Our central hypothesis is that PHB nitrosylation is critical for its neuroprotective function and that disturbances of PHB nitrosylation is detrimental. We will use a mutant knockin mouse, in which the sole cysteine residue of PHB protein is mutated so that PHB cannot be nitrosylated, and a PHB neuronal knockout mouse, in which PHB deletion is complemented by AAV expressing wild type or non-nitrosylated C69S mutant to analyze the mechanisms of NO regulation and the effects of loss of PHB nitrosylation on PHB function, in IPC. Three specific aims will systematically test the hypothesis. The results of the proposed studies will reveal a previously undescribed regulatory mechanism of PHB and could benefit patients at risk of stroke and other neurological conditions.